1. Project Summary/Abstract Hypoxia (low oxygen) is a known key driver of tumor aggressiveness and tumor resistance to radiation treatment (RT) in a variety of solid cancers1-8. Given that about 50% of cancer patients receive RT (~800,000 cases per year in the US), Omniox has developed a novel oxygen carrier protein, OMX, which is engineered to specifically deliver oxygen to low oxygen regions of tumors and enhance RT efficacy. Omniox is supported by NCI SBIR (Phase I, II, IIb) and Wellcome Trust translational awards to test its lead drug candidate as an adjuvant to standard-of-care RT for the treatment of newly diagnosed glioblastoma (GB) patients. Omniox has received FDA pre-IND support for, and is currently conducting, IND-enabling manufacturing and GLP toxicology studies for the first-in-human clinical trial scheduled at the University of California, San Francisco (UCSF) for 2017. Importantly, non-invasive PET imaging with hypoxia-selective agents has shown that hypoxic volume in GB tumors is strongly associated with rapid disease progression and poor survival1 due to its blunting effects on standard-of-care RT. Previous efforts to oxygenate tumors in human clinical trials have shown ambiguous results due in part to heterogeneity in tumor hypoxic burden and the lack of targeted approach for patient selection13. Therefore, the goal of this proposal is to develop predictive non-invasive PET imaging biomarkers that will identify the GB patient population that can substantially benefit from OMX therapy. Omniox and the UC Davis School of Veterinary Medicine completed a Phase 0 canine clinical trial to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of OMX in canine patients presenting with brain tumors. As part of the trial, Omniox partnered with Brain Biosciences to use their PET scanner designed specifically for brain imaging (CerePET?). Omniox and the key collaborators on this AIP grant, Drs. Simon Cherry (Aim 1) and Allison Zwingenberger (Aim 2) at UC Davis, and David Beylin of Brain Biosciences (Aims 1 and 2) are developing protocols for PET imaging of canine brain patients, with successful initial scans already generated. In this proposal, we will build on our positive preliminary results in rodents and canines to evaluate imaging of tumor hypoxia and OMX tumor accumulation as predictive biomarkers of OMX activity in an intracranial rat GB model and in canine glioma patients. These data will then be used to inform an IND application and a human biomarker clinical trial. Developed from the thermostable H-NOX protein family discovered at UC Berkeley, OMX is a trimerized, PEGylated H-NOX oxygen (O2)-binding variant engineered to diffuse deep into hypoxic tumor tissue commonly associated with leaky blood vessels. The H-NOX moiety has been engineered to have a high affinity for O2 whereby it retains O2 in normoxic tissue and specifically releases it in regions of severe hypoxia. Omniox has completed a series of preclinical studies that support this proposal: 1) OMX is biochemically well-characterized and is currently being manufactured at high yields and purity for GLP toxicology and human GB clinical trials 2) OMX has been safely administered to over a thousand mice and rats, several healthy Beagle dogs, globally hypoxic lambs, and fifteen canine brain cancer patients from our canine clinical trial (Table 1) 3) OMX extravasates across the leaky vasculature and accumulates in hypoxic tumor tissue as seen in: a) mouse and rat intracranial tumors using immunohistochemistry (IHC) (Fig. 1) and ELISA (data not shown); b) tumor bearing mice and rats using PET imaging of 89Zr-labeled OMX (Fig. 5); and c) in tumors of canine brain cancer patients as seen by IHC and ELISA (Fig. 6) 4) OMX administration oxygenates tumors as demonstrated by: a) the direct measurement of increased oxygen partial pressure (pO2 ) using an OxyliteTM probe inserted into hypoxic mouse tumor tissue (Fig. 2); downregulation of hypoxia-regulated transcription factors and downstream signaling as measured by IHC, FACS,(data not shown), and ELISA (Fig. 3); and c) hypoxia reduction in rat intracranial tumors as measured by 18F-FMISO signal, a PET hypoxia tracer, in a pilot imaging study (Fig. 5). 5) Mice bearing subcutaneous tumors treated with OMX prior to RT showed significant delays in tumor growth and a 50% cure rate compared to mice treated with RT alone (Fig. 4) 6) CerePET can be used to image rat and canine brains with high resolution (Fig. 7) In this proposal, we present an innovative translational strategy to develop and preclinically test a PET imaging companion diagnostic that can potentially be used as a predictive marker of drug candidate efficacy in future human clinical trials. We will build on our existing results in intracranial rat and mouse tumor models and canine glioma patients to optimize and validate a non-invasive clinically relevant imaging method that, if confirmed in human clinical studies, will enable selection of a targeted patient population whose RT treatment is likely to be substantially enhanced by OMX-mediated tumor oxygenation. 2016 Omniox, Inc. CONFIDENTIAL ? Not for Distribution